A multi-part research program has been designed to yield information about the expression of the transfer RNA genes in E. coli, the biosynthesis and role of modified nucleotides in E. coli tRNA, and the organization and expression of the genes which code for the metabolically stable E. coli 4.5S and 6S RNAs. The biological activities of 16 undermodified forms of phenylalanine tRNA will be with the fully modified species in an effort to reveal the role of tRNA base modification in the cell. To elucidate the mechanism of dihydrouridine biosynthesis in tRNA, a dihydrouridylate synthetase will be enriched and characterized using as substrate, dihydrouridine-deficient tRNA from amino acid-starved relaxed control E. coli. In an effort to identify tRNA species whose synthesis and rate of turnover are regulated differently from that of bulk tRNA, a two-dimensional acrylamide gel electrophoresis procedure will be used to determine the levels of individual tRNA species in cells in a variety of metabolic states. The fine structure and transcriptional properties of the five-cistron operon for the major E. coli leucine tRNA will be determined from in vitro transcription experiments in which DNA enriched for this particular operon is used as template material. The chromosomal distribution of repeated DNA sequences complementary to 4.5S and 6S RNAs will be determined by analyzing RNA:DNA hybrids constructed from DNA fragments produced with specific restriction endonucleases. Finally, to determine whether all/most of the repeated DNA sequences complementary to the E. coli 4.5S and 6S RNAs are actually expressed in vivo, in vivo RNA synthesis experiments will be performed with various partial diploid strains of E. coli which contain elevated amounts of the different 4.5S and 6S DNA sequences. The transcriptional activity of the individual 4.5S and 6S DNA sequences will be estimated by comparing the amount of 4.5S/6S RNA formed with the cellular content of 4.5S/6S DNA.